Deciphering the pathogenic role of a variant with uncertain significance for short QT and Brugada syndromes using gene‐edited human‐induced pluripotent stem cell‐derived cardiomyocytes and preclinical drug screening

In the present study, we show that the Calcium Voltage-Gated Channel Auxiliary Subunit Beta 2 (CACNB2) variant c.1439C > T/p.S480L is linked to the clinical phenotype of short QT syndrome 5 (SQTS5) overlapped with Brugada syndrome (BrS). The PI3K pathway may contribute to the arrhythmogenesis of the disease. PI3K-activator and amiodarone but not sotalol may be effective drugs for treating arrhythmias in SQTS5-patients carrying but not limited to this variant. cells a SQTS5-patient overlapped with CACNB2 study significance of the variant for the clinical phenotype by combin-ing gene and electrophysiological in the

Dear Editor, In the present study, we show that the Calcium Voltage-Gated Channel Auxiliary Subunit Beta 2 (CACNB2) variant c.1439C>T/p.S480L is linked to the clinical phenotype of short QT syndrome 5 (SQTS5) overlapped with Brugada syndrome (BrS). The PI3K pathway may contribute to the arrhythmogenesis of the disease. PI3K-activator and amiodarone but not sotalol may be effective drugs for treating arrhythmias in SQTS5-patients carrying but not limited to this variant.
The SQTS is characterized by a shortening of the corrected QT (QTc) interval, which has been linked to sudden cardiac death. 1,2 Implantable cardioverter-defibrillator therapy is associated with numerous complications. 3 Therefore, drug therapy is important to optimize the treatment of SQTS patients. Recently published data have reported that hydroquinidine is effective in prolonging the QTc interval in SQTS patients. Notably, data have shown that hydroquinidine might exert different effects depending on the genetic variant and/or SQTS form. 4,5 Based on the limited evidence of the clear role of variants in calcium channel subunits in SQTS and the absence of alternative therapies in this rare cohort, 6 we aimed to use cardiomyocytes from induced pluripotent stem cells (hiPSC-CMs) derived from a SQTS5-patient overlapped with BrS carrying a variant in CACNB2 to study the significance of the variant for the clinical phenotype by combining gene editing and electrophysiological analysis in order to identify possible effective drugs for the disease. HiPSC-CMs offered advantages over other models to model channelopathies in the dish. 7,8 For this study, human iPSC lines from one SQTS patient, from two healthy donors, and two Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene-edited hiPSC lines were used ( Figure S1A Figure S2).
The SQTS patient showed an abbreviated QTc interval with a QTc of 330 ms ( Figure 1A) and was admitted due to an aborted cardiac arrest. A genetic screening of SQTS related genes of this patient detected a variant, namely c.1439C>T/p.S480L (dbSNP rs121917812; Clinvar RCV000010155.3; NM_000724.4: c.1439C>T; NM_201590.3: c.1442C>T) in CACNB2, a beta-subunit of L-type calcium channel ( Figure 1C). Sequencing of SQTS related genes confirmed the existence of the same variant in other first or second-degree relatives ( Figure 1B,C) with a SQTS and BrS phenotype.
The durations of AP at 50% (APD50) and 90% repolarization (APD90) were significantly shorter in SQTS5 cells compared to that in the cells from the healthy donor and isogenic control cells ( Figure 1D-F). The maximal depolarization speed (V max ) of APs was decreased ( Figure 1G). Of note, the APD-shortening remained at all the tested frequencies. In addition, HD-MEA recordings detected that the field potential duration in SQTS5-hiPSC-CMs was also significantly shorter compared to the healthy or isogenic control ( Figure 1H-I). In recordings of spontaneous APs, the frequency of cell beating in SQTS5-hiPSC-CMs was significantly slower than that in healthy cells, without difference in maximal diastolic potential ( Figure S3C,D,G,H). After the APD parameters were corrected for beating frequency (Bazettťs correction), the corrected APDs were also shorter in SQTS5-hiPSC-CMs ( Figure S3C-F).
The L-type calcium channel current (I Ca-L ) was significantly decreased in SQTS5-hiPSC-CMs as compared to that from the healthy control and isogenic control ( in isogenic control hiPSC-CMs. Besides, the inactivation curve shifted to more negative potentials, and recovery from inactivation decelerated in SQTS5-hiPSC-CMs compared with both healthy and isogenic control cells (Figure 2C-H). Western blot and immunostaining analyses detected that the protein expression level of CACNB2 was significantly decreased in SQTS5-hiPSC-CMs ( Figure S4). The alpha subunit of L-type Ca 2+ channel CACNA1C was slightly increased in cell lysates ( Figure S4) but not in the cell membrane ( Figure S4).
Since loss-of-function of the sodium channel is a main feature of BrS, the peak sodium channel current (I Na ) and their kinetics were assessed ( Figure 3A,B and Figure S5A-F). In SQTS5-hiPSC-CMs, the peak I Na and activation were significantly suppressed compared to healthy cells.
To test the arrhythmogenicity of hiPSC-CMs of the SQTS5-patient, spontaneous calcium transients and spontaneous APs were recorded. Compared to healthy donor and isogenic control cells, a higher number of SQTS5-cells  Figure 3C-F). The interval variability (standard deviation of beating intervals) in SQTS5-hiPSC-CMs was larger than that in donor and isogenic control cells ( Figure 3C-F).
For details about methods, please see Supporting Information. As expected, insertion of the present CACNB2 variant using CRISPR/Cas 9 in a further control cell line ( Figure S6) led to a loss-of-function of I Ca-L and an APDshortening with a reduction of V max ( Figure S7).
Quinidine and amiodarone prolonged APD (Figures S8 and S9) but only amiodarone showed a significant antiarrhythmic effect ( Figure S9). Sotalol showed no effect in SQTS5-cells although it prolonged APD in healthy cells (Figures S10 and  S11).
Since the PI3K pathway may influence ion channel function and QT interval, 9,10 we measured the protein levels of PI3K and Akt. Both total and phosphorylation levels of Akt and PI3K were decreased in SQTS5-hiPSC-CMs compared to healthy cardiomyocytes ( Figure 4A-C). Whereas a PI3Kactivator (IGF-1, 100 ng/ml) reduced the interval variability and arrhythmic events, a PI3K blocker (alpelisib, 5 μM) enhanced both ( Figure 4D-F). In addition, the PI3Kactivator prolonged the APD (Figure 4G,H). In conclusion, the variant c.1439C>T/p.S480L in the CACNB2 gene is responsible for phenotypic changes of SQTS5 overlapping with BrS. Amiodarone reduces arrhythmic events in the dish and based on this, we may assume that it may be clinically more effective than quinidine for treating SQTS5 and /or BrS. The PI3K/Akt activity can be reduced in SQTS5 and an activator of this signaling pathway may rescue the phenotype.  ) and presence of alpelisib (PI3K inh.) or IGF-1 (PI3K act.). (G) Mean values of action potential duration at 50% repolarization (APD50) in the absence (SQTS5, Healthy) and presence of IGF-1 (SQTS5 + PI3K act. and Healthy + PI3K act.) (H) Mean values of action potential duration at 90% repolarization (APD90) in the absence (SQTS5, Healthy) and presence of IGF-1 (SQTS5 + PI3K act. and Healthy + PI3K act.). "n" represents the number of cells. *p < .05 versus SQTS5 according to one-way analysis of variance (ANOVA) with Holm-Sidak post-test